Human coronaviruses (HCoV)-OC43 and HCoV-229E have long been known as agents of the common cold. The emergence of SARS-CoV as the etiological agent of severe acute respiratory syndrome in 2003 and more recently, Middle East respiratory syndrome (MERS)-CoV with 10% and 30% mortality respectively, make it imperative to understand the coronavirus-host interactions that contribute to virulence. The ability of viruses to evade or antagonize the type I interferon (IFN) response plays a vital role i pathogenesis. An understudied aspect of immune evasion by coronaviruses is the antagonism of the oligoadenylate synthetase (OAS)- ribonuclease (RNase) L pathway, an IFN-induced potent antiviral mechanism. Once activated by double- stranded RNA, OAS synthesizes 2',5'-linked oligoadenylates (2-5A) that activate RNase L. RNase L cleaves single-stranded RNA leading to degradation of viral genomes and host RNA, protein synthesis arrest, and apoptosis. The group 2a Betacoronavirus, mouse hepatitis virus (MHV) accessory protein ns2 is a 2',5'- phosphodiesterase (PDE), which cleaves 2-5A thereby preventing activation of RNase L. PDE activity is a critical determinant of replication in Kupffer cells and liver sinusoidal endothelialcells as well as hepatovirulence in mice. Other group 2a Betacoronaviruses, including HCoV-OC43, encode ns2 homologs that we have recently confirmed as RNase L antagonists. Additionally, the group 2c Betacoronavirus MERS-CoV accessory protein NS4b is predicted to be a PDE and in preliminary experiments can degrade 2-5A. These findings lead to the hypotheses that group 2c as well as group 2a Betacoronaviruses antagonize the OAS-RNase L pathway by PDE-mediated cleavage of 2-5A and that OAS-RNase L antagonism contributes to human Betacoronavirus replication in the respiratory tract, and potentially to pathogenesis. Aim 1 will investigate the predicted PDE activity of MERS-CoV NS4b and related group 2c Bat- CoV homologs. PDE activity of recombinant NS4b and homologs will be assessed in vitro by a 2-5A cleavage assay and in transfected cells by measuring ribosomal RNA integrity and quantifying 2-5A levels. In addition, chimeric MHVs expressing inactive ns2 and each active NS4b PDE will be constructed and the rescue of replication and hepatitis assessed. Finally, the impact of the nuclear localization, signal located on NS4b and not present on the group 2a Betacoronavirus PDEs, will be assessed. Aim 2 will examine the contribution of RNase L antagonism to the success of HCoV-OC43 and MERS-CoV replication in primary human airway cells including epithelial, endothelial and macrophages. Metrics for analysis of antagonism will include viral titers and ribosomal RNA integrity. Results will be correlated with basal expression levels of OAS genes in these cell types. This study will contribute to the understanding of coronavirus-host interactions, in particular the impact of PDE-mediated RNase L antagonism on human respiratory coronavirus infection. Finally, virus-encoded PDEs potentially provide a tantalizing target for antiviral therapeutics.